Chromosomes, Nuclear Genes and the Phylogenetic Placement Within the Reptilia of Sphenodon (Tuatara)

Chromosomes were examined from five populations of Sphenodon (tuatara)  using giemsa, Ag-NOR, C-, G- and RE- banding. There were no differences between species, populations or sexes, although one animal had a structural heteromorphism. Chromosome morphology homology to Testudines (turtles), Aves (birds) and to a lesser extent Crocodylia (crocodiles) allowed reconstruction ofa Reptilian proto-karyotype, dated to 300 million years ago. DNA sequence was isolated from the WT1, AMH, DMRT1, FoxG1 and 28S. No variation was present in Sphenodon 28S, FoxG1 or AMH sequence. 28S could be dated to a common ancestor with Testudines, similar to the archaic karyotype. FoxG1 and AMH reflect an Oligocene divergence, WT1 divides north-eastern North Island and Cook Strait, and can be dated to the Pleistocene or the Pliocene, and DMRT1appears a recent post- pliocene divergence. FISH localised DIG-labelled probes of AMH to chromosome 11 and WT1 to chromosome 13 or 14. Human telomeric probes localised to Sphenodon telomeric regions demonstrating the highly conserved nature of telomeric sequences. Comparative genomic hybridisation with chicken chromosomes did not produce any regions of homology, implying significant chromosomal and DNA changes since the Orders shared a common ancestor, although macrochromosome morphology has remained similar. Sphenodon chromosomal and nuclear DNA analyses demonstrate evolutionary decoupling, supporting recent mtDNA work.